**5. Targets and techniques for detection of insecticide resistance:**

#### **5.1 Metabolic resistance**

It is the most common mechanism of development of resistance to insecticides. In this type of resistance, either the enzymes which detoxify the insecticide are over expressed or there is an altered affinity of the enzyme for the compound used, mainly caused by substitution of amino acids, mainly in the three major enzyme families (cyrochrome P450 monooxygenase, glutathione S-transferase [GSTs] and esterase) which are involved in metabolism of the insecticide compound [41].

In mosquito species, the vectors of malaria and lymphatic filariasis, several CYP450 genes has been documented to be involved in resistance to pyrethroids, *viz.,* CYP6P3 and CYP6M2, CYP6P9a, CYP6Z3 [42], while CYP6Z1 is attributed for conferring resistance to both carbamates and pyrethroids [43]. CYP9M10 and CYP6AA7 confer resistance to the insecticide permethrin in *Culex* species [44, 45]. The GSTs confer resistance to DDT in mosquitoes, i.e., *Anopheles*, and GSTe2 GSTe3, GSTe4 have been reported to be involved in pyrethroid resistance in *An. funestus* in Uganda and Kenya [46]. Several P450s and GST genes were reported to be overexpressed in a deltamethrin-resistant *An. sinensis* in China and Southeast Asian countries [47]. In a recent study carried out by Yan et al., several genes have been identified which confer permethrin resistance to *Culex pipiens quinquefasciatus,* particularly, 2 CCEs, 6 GSTs, and 7 P450s gene were highly expressed [49].

*Insecticide Resistance in Vectors of Medically Important Parasitic Infections DOI: http://dx.doi.org/10.5772/intechopen.100583*

The detection of the enzymatic activity in biochemical assays is the most commonly used method to assess development of resistance to insecticides in insects. In several studies done to detect pyrethroid resistance, increased P450 monooxygenases and esterase activity have been recorded in the resistant strains of *Anopheles*, *Culex*, and triatomine bugs [40]. For sandfly, metabolic resistance is not adequately studied and only some studies of insecticide resistance related to bioassays on *P. argentipes* and *P. papatasi* populations from India and on *Lutzomyia* populations in South America have been reported [29].

#### **5.2 Target site resistance**

Here, the target site of the action of the insecticide may be modified genetically. As a result, the binding or interaction of the insecticide at its site of action is thereby prevented which decreases the efficacy of the insecticide.


#### **Table 2.**

*Genetic determinants and point mutations associated with insecticide resistance in various insect vectors [18, 40, 43, 45, 46, 48–58].*

Various target-site mutations in the voltage-gated sodium channel (*VGSC*) gene were recorded in response to pyrethroids in different species of mosquitoes, *viz*., *Anopheles gambiae* complex, *An. funestus* and *An. culicifacies*, *Phlebotomus argentipes* and triatomine bugs (**Table 2**). The most common resistance associated mutation reported in the AChE gene is G119S in species of *Anopheles* and *Phlebotomus*.

### **6. Methods of detection of insecticide resistance**

The development of resistance to insecticides in insects is a complex phenomenon which depends on many direct and indirect factors. The direct factors include the natural variations in the genetic, biochemical, physiological, ecological and behavior of insects, while the indirect factors are the operational factors such as categories of insecticides used, the timing and method used for their application [59]. There are different bioassays which exploit some of these factors and help in detecting insecticide resistance, i.e., by phenotypic, genotypic and proteomic analysis.

The laboratory bioassays are useful in detecting susceptibility, tolerance and resistance in vectors against insecticides [60]. The phenotypes of vectors are utilized in detecting the insecticide resistance in methods such as by bottle bioassays. In this, a range of concentrations of the insecticide is used to study the target site resistance followed by knocking certain genes in the insect population [61]. The WHO cone tests, wireball assays, tube tests [62] and the Centers for Disease Control and Prevention (CDC) bottle bioassays take long exposure times, therefore, an alternative method – mosquito contamination device (MCD) bottle bioassay – has been developed for the control of malarial vectors in resource poor settings [63]. In vector control programmes, it is recommended that bottle bioassays should be routinely use in laboratory to measure the phenotypic resistance of a vector against a particular insecticide so as to determine whether it is still effective [64].

In addition to the traditional bioassays for determining resistance to insecticides, currently, various molecular markers and techniques to detect target site mutations have been developed. These genetic mutations can be identified by various PCR based methods such as random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), microsatellites and single nucleotide polymorphisms (SNP). To observe changes at the RNA level, molecular techniques such as real-time polymerase chain reaction (RT-PCR), differential display reverse transcription PCR, northern blot and microarrays may be used. For protein estimation enzyme assays, various techniques such as enzyme linked enzyme-linked immunosorbent assay (ELISA), western blot, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) may be used [65, 66]. In addition to this, to study the mechanism of resistance at the molecular level through proteomics, the identification of resistance-related proteins with their expression profiling can provide knowledge of the activity of proteins related to insecticide resistance in insects [67].

#### **6.1 Detection of insecticide resistance in malaria vectors**

#### *6.1.1 Phenotypic assays*

The WHO and CDC bioassays are the most common assays carried out to detect the insecticide resistance in malarial vectors till now. The WHO bioassays have been carried out in various studies, such as in detection of *Anopheles culicifacies* resistance to DDT, malathion and deltamethrin in India (60), *An. stephensi* in eastern

#### *Insecticide Resistance in Vectors of Medically Important Parasitic Infections DOI: http://dx.doi.org/10.5772/intechopen.100583*

Ethiopia [68], and *An. arabiensis* susceptibility to bendiocarb, lambda-cyhalothrin and deltamethrin in Yemen [69]. The CDC bottle bioassay had been carried out to detect metabolic resistance as well as biochemical resistance against permethrin in *An. arabiensis* in Tanzania [70], and to quantify the resistance of insecticides of deltamethrin, lambda-cyhalothrin, alpha-cypermethrin, permethrin and DDT in *An. darlingi, An. nuneztovari* and *An. albimanus* in Colombia [71]. Another bioassay used for detecting the phenotypic resistance in the malarial vectors is MCD bottle bioassay. These bioassays are helpful and significant as they detected the effect on the behavior of *An. stephensi* and *An. gambiae* after the exposure to insecticides [63].
